Panel A shows a cartoon representation of the structural model. The model includes zoomed-in views highlighting residues at the protein binding interface, with dotted lines indicating potential electrostatic interactions. Panel B presents three isothermal titration calorimetry (ITC) experiments. Each graph is a line plot showing the differential power (DP) in microwatts over time in minutes, and the enthalpy change (delta H) in kilojoules per mole against the molar ratio. The first graph shows a clear interaction with a dissociation constant of 2.54 plus minus 0.302 micromolar, while the other two graphs show no interaction. Panel C displays western blot results from pull-down assays. The blots show protein bands for Flag-tagged Ana1 mutants and GFP-tagged Cep135, with labels indicating molecular weight in kilodaltons and specific mutants.
Identification of key residues within Ana1 that bind Cep135. (A) Cartoon representation for the structural model of the Cep135250–352-Ana1276–298 complex predicted by AF3-based analysis. Underneath are the zoomed-in views showing residues at the protein-binding interface. Dotted lines indicate potential electrostatic interactions formed between conserved residues within Ana1 and Cep135. (B) ITC experiments showing substitution of the conserved, core hydrophobic residues at the binding interface with negatively charged Asp residues completely abolished the Cep135-Ana1 interaction. The experiment was repeated three times, and the average Kd value is displayed. (C) S2 cells were transiently cotransfected with Flag-tagged Ana1 mutants and GFP-tagged Cep135. Extracts were then subjected to pull-down assays using GFP beads and were analyzed by western blotting. The experiment was repeated three times with similar results. Source data are available for this figure: SourceData F4.